Molecular studies on soluble acid invertases in the potato (Solanum tuberosum L.) tubers

Abstract

Sucrose is a disaccharide composed of glucose and fructose, i.e. Glc (12) Fru or Fru (21) Glc. This nonreducing sugar is a major intermediate product of photosynthesis. Sucrose synthesis occurs exclusively in the cytosol of photosynthetic cells. In many plants sucrose represents the most common form of sugar found in translocation stream where it is transported from leaves (source) to nonphotosynthetic tissues (sink) such as flower, fruit, seed, and root. Sucrose not only plays a vital role in plant growth and development but is also a leading commercial commodity. Sucrose and hexoses also play signaling roles in regulating gene expression and plant development. The cleavage reaction of sucrose is catalyzed by two enzymes in higher plants: Sucrose synthase (Sus, EC 2.4.1.13) or invertase (INV, EC 3.2.1.26). Sus degrades sucrose in the presence of UDP into UDP-glucose and fructose, where as the INV hydrolyses sucrose into glucose and fructose. Based on their solubility, subcellular localization, pH optima and isoelectric point, three different types of invertase isoenzymes can be distinguished: i) vacuolar invertase (VIN)-most plant species contain at least two isoforms of vacuolar invertase; ii) cell wall invertase (CWIN)-several isoforms of extracellular invertase ionically bound to the cell wall; iii) neutral invertases-also known as cytoplasmic invertases (CIN). CWIN is known to play crucial role during flower, seed, and fruit development; whereas VIN as a key modulator for hexose accumulation and cell expansion, but the role of CIN in plants is still unclear. Potato is a very important member of the Solanaceae family. Currently, potato is the third most important food crop which comes only after wheat and rice. Storage of potato tubers at low temperatures (~ 40C) for several weeks leads to breakdown of starch into sugars in tubers with consequent accumulation of reducing sugars-a phenomenon known as ‘cold-induced sweetening’ which negatively affects the quality of potato chips and French fries. It is commonly believed that in potato, invertases together with other proteins are involved in the ‘cold sweetening’ process. Therefore, inhibition of invertase (vacuolar invertase in particular) gene function is an important aspect of applied research. There is no report available on invertases from the Indian potato cultivars at biochemical, genetic, and molecular levels. In this study, two cDNA clones designated as AI-01 and AI-02 (2013 bp and 1945 bp, respectively with 99% sequence identity) encoding vacuolar invertase isoforms were isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR approach. The corresponding predicted proteins consisted of 635 amino acids (designated as KC-VIN1, lacking a few amino acids at N-terminus) and 639 amino acids (designated as KC-VIN2), respectively. They showed 99% identity, and found to vary at several locations with mostly nonconservative substitutions. Multiple sequence alignment of vacuolar invertase homologs covering four Solanaceae family members revealed some notable distinguishing sequence features (signature-type sequences). A consensus sequence was predicted using 45 vacuolar invertase sequences from 27 taxonomically different plant species, and a phylogenetic tree was generated to know the evolutionary relatedness between them. Hydrophobic characters were predicted, and compared in different plant species. The levels of reducing sugars were determined in the potato tubers from the different cultivars during storage at ~40C for 4 to 8 weeks. As a preliminary study, vacuolar invertase expression patterns in the tubers of some Indian potato cultivars were analyzed by semi-quantitative RT-PCR and extractable enzyme assay. In all the potato cultivars, the overall expression level of invertase was found to be considerably higher after storage at low temperature as compared to the freshly harvested tubers. All the data suggest that the potato cultivars under study belong to type 1 category. For inhibition of vacuolar invertase gene function, the following strategies were adopted. The constitutive (the CaMV 35S promoter) and tuber-specific (the GBSSI promoter) promoters were used for making a series of antisense and sense binary genetic constructs using different regions of the vacuolar invertase cDNAs, AI-01 and AI-02. Kufri Chipsona-1 a processing potato cultivar) was used during Agrobacterium-mediated genetic transformation. Minitubers were produced from the transgenic potato lines corresponding to the individual binary constructs. After harvesting, the mini tubers were kept at room temperature for 2 to 3 weeks and then transferred to different temperatures to carry out biochemical analysis. Soluble sugars were extracted from mini tubers stored at different temperatures i.e. 4ºC, and 25ºC for four weeks and eight weeks. In control (non transgenic) potato tubers, the reducing sugar level was found to be in the range of 3.80-4.20 mg g-1 FW. Transgenic potato tubers corresponding to different transgenic lines showed significantly (P<0.05) lower accumulation of reducing sugars in the range 0.66-1.75 mg g-1FW at 40C. The level of reducing sugars was reduced by 50-80% in the trangenic potato tubers as compared to control. The activity of vacuolar invertase was found to be ~1.60 nmol (min.mg)-1 in control potato tubers after storage for four weeks at 4ºC; whereas, the values for a number of transgenic tubers were considerably low; found to be in the range of 0.058-0.216 nmol (min.mg)-1. All these data are provided categorically in the thesis. A few observations were made based on sugar data analyses: a) the independent transgenic lines corresponding to the individual genetic constructs showed variations in the reducing sugar level during low temperatures storage, b) reducing sugar level was found to be comparable in some of the transgenic lines generated by both antisense and sense binary genetic constructs, c) the constitutive CaMV 35S and the tuber-specific GBSSI promoter appeared to exhibit more or less similar effects, and d) use of invertase cDNA fragment towards the 3′ terminus in the binary genetic constructs in either orientation appeared to be more effective. For inhibition of invertase gene expression in potato, different molecular techniques were adopted by other laboratories such as antisense approach, expression of invertase inhibitors, RNAi approach for post transcriptional gene silencing (PTGS). By such approaches transgenic potato lines with desirable traits were generated. Some of the transgenic potato lines as generated in this study are quite promising in terms of repression of cold-induced sweetening. In conclusion, all the data as generated and presented in the thesis are important and relevant with regard to both basic and applied aspects.